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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2012-11-01 to 2012-11-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD test guideline No. 471 and in compliance with GLP
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP (Inspected on 2012-06-20)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
- Name of test material (as cited in study report): Ethylene Brassylate
- Physical state: colourless/pale yellow liquid
- Storage condition of test material: Room temperature, dry.

Method

Target gene:
Histidine gene for S. typhimurium and tryptophan gene for E.coli
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (S9 fraction, prepared from male Sprague-Dawley derived rats, dosed i.p. with phenobarbital sodium and 5,6-benzoflavone)
Test concentrations with justification for top dose:
Test 1: 5, 15, 50, 150, 500, 1000 and 5000 µg/plate.
Test 2: 50, 150, 500, 1000 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:DMSO
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at 50 mg/mL in solubility checks performed in-house.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
(See Table 7.2.1/1)
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
In the absence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
(See Table 7.2.1/1)
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
In the presence of S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1 in agar (plate incorporation), Experiment 2 preincubation

DURATION
- Preincubation period (Exp 2): 30 minutes at 37°C.
- Exposure duration: ca. 72 hours at 37°C.

NUMBER OF REPLICATIONS: triplicate plates per dose level

DETERMINATION OF CYTOTOXICITY
- Method: growth assessment of the bacterial background lawn

OTHER: ACCEPTANCE CRITERIA:
For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the current historical control range for the laboratory unless otherwise justified by the Study Director. The historical range is maintained as a rolling record over a maximum of five years. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537, which have relatively low spontaneous reversion rates) that of the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 10E09/mL.
Evaluation criteria:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system. No statistical analysis is performed.
If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. Biological importance will be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgement.
Statistics:
The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
weak toxicity at 5000 µg/plate without S-9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Effects of pH: not applicable
- Effects of osmolality: not applicable
- Evaporation from medium: the test material is not classified as a VOC.
- Water solubility: the test material was solubilised in DMSO to improve solubility.
- Precipitation: none
- Other confounding effects: none

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory. Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- First test:
Evidence of weak toxicity, observed as a slight reduction in revertant colony numbers, was obtained in the Salmonella strains following exposure to Ethylene Brassylate at 5000 µg/plate in the absence of S9 mix. No evidence of toxicity was obtained in the E. coli strain, or in the presence of S9 mix. A maximum exposure concentration of 5000 µg/plate was, therefore, selected for use in the second test.
- Second test:
Evidence of weak toxicity, observed as a slight reduction in revertant colony numbers, was obtained in the Salmonella strains following exposure to Ethylene Brassylate at 5000 µg/plate in the absence of S9 mix. No evidence of toxicity was obtained in the E. coli strain, or in the presence of S9 mix.

OTHER:
The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test substance formulation.
The viability counts confirmed that the viable cell density of the cultures of the individual organisms exceeded 109/mL in all cases, and therefore met the acceptance criteria.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Ethylene Brassylate is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537, TA98, TA100, and E.coli WP2 uvrA according to the criteria of the Annex VI of the of the Regulation (EC) No 1272/2008 (CLP) and of the Directive 67/548/EEC.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E.coli strain WP2 uvrA were exposed to Ethylene Brassylate diluted in DMSO both in the presence and absence of metabolic activation system (10% v/v S9 fraction in standard co-factors) using both the Ames plate incorporation and pre-incubation methods, in triplicate. Concentrations of Ethylene Brassylate up to, and including, 5000 µg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration.

Evidence of weak toxicity, observed as a slight reduction in revertant colony numbers, was obtained in the Salmonella strains following exposure to the test material at 5000 µg/plate in the absence of S9 mix. No evidence of toxicity was obtained in the E. coli strain, or in the presence of S9 mix.

No evidence of mutagenic activity was seen at any concentration of Ethylene Brassylate in either mutation test.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

Under the test condition, Ethylene Brassylate is not mutagenic in the presence and absence of metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA according to the criteria of the Annex VI of the of the Regulation (EC) No. 1272/2008 (CLP) and of the Directive 67/548/EEC.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.